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Use of Sera from Humans and Dolphins with Lacaziosis and Sera from Experimentally Infected Mice for Western Blot Analyses of Lacazia loboi Antigens

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Antibodies in the sera of patients with lacaziosis recognized an approximately 193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.
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CLINICAL AND VACCINE IMMUNOLOGY, Jan. 2008, p. 164–167 Vol. 15, No. 1
1556-6811/08/$08.000 doi:10.1128/CVI.00201-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
NOTES
Use of Sera from Humans and Dolphins with Lacaziosis and Sera from
Experimentally Infected Mice for Western Blot Analyses of
Lacazia loboi Antigens
Leonel Mendoza,
1
* Andre´a F. F. Belone,
2
Raquel Vilela,
1
Manuela Rehtanz,
3
Gregory D. Bossart,
4
John S. Reif,
5
Patricia A. Fair,
6
Wendy N. Durden,
7
Judy St. Leger,
8
Luiz R. Travassos,
9
and Patricia S. Rosa
2
Biomedical Laboratory Diagnostics Program, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing,
Michigan 48824-1031
1
; Instituto Lauro de Souza Lima, Bauru, Sa˜o Paulo, Brazil
2
; New York University, Department of Basic Sciences,
Infectious Diseases, New York, New York 10010
3
; Harbor Branch Oceanographic Institution, Center for Coastal Research,
Marine Mammal Research and Conservation, Fort Pierce, Florida 34946
4
; Department of Environmental and
Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523
5
; National Oceanic and
Atmospheric Administration/National Ocean Service/Center for Coastal Environmental Health and
Biomolecular Research, Charleston, South Carolina 29412
6
; Hubbs-SeaWorld Research Institute,
Orlando, Florida 32821-8043
7
; Department of Pathology, SeaWorld, San Diego,
California 92109
8
; and Department of Microbiology, Immunology and
Parasitology, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil
9
Received 14 May 2007/Returned for modification 5 August 2007/Accepted 16 October 2007
Antibodies in the sera of patients with lacaziosis recognized an 193-kDa antigen and other Lacazia loboi
antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect
a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral
response to L. loboi antigens by using multiple host sera.
Lacazia loboi is an uncultivated fungal pathogen predomi-
nantly restricted to Latin America. Lacaziosis has been re-
corded among humans dwelling in this geographical area, tour-
ists visiting areas of endemicity (4, 10), and workers handling
infected dolphins (19, 24). Lacaziosis has also been diagnosed
in dolphins inhabiting the coastal areas of the United States (9,
12, 19), Brazil (16), and France (25). Using molecular meth-
ods, the phylogenetic features of L. loboi were only recently
deciphered (13, 18, 31). These studies placed L. loboi as a sister
group to Paracoccidioides brasiliensis and, in turn, linked to the
other dimorphic members of the Onygenales in the family
Ajellomycetaceae.
The clinical features of Lacazia loboi and P. brasiliensis in-
fections are different, but both fungi develop yeast cells, which
are difficult to differentiate microscopically, in the host’s in-
fected tissues (7). This resemblance was used by some inves-
tigators to classify this pathogen in the genus Paracoccidioides
(1, 6, 8). Thus, due to L. loboi’s inability to grow in culture,
immunological studies have traditionally been carried out with
antigens extracted from mycelial cultures of P. brasiliensis (15,
17, 21, 23–27, 30). The objective of our study was to charac-
terize the immunogenic proteins extracted from L. loboi yeast-
like cells in Western blotting analyses, using sera from humans
and dolphins with lacaziosis and sera from experimentally in-
fected mice.
Sera from four human patients with lacaziosis from Acre
State, Brazil, and from one Brazilian patient with proven para-
coccidioidomycosis and four sera from experimentally L. loboi-
infected mice were collected and stored at 80°C until use (2).
Sera from three bottlenose dolphins (Tursiops truncatus) in-
fected with L. loboi were evaluated: one wild dolphin infected
with L. loboi was captured in the Indian River Lagoon, FL, one
was a stranded Indian River Lagoon bottlenose dolphin, and
one was a captive bottlenose dolphin from SeaWorld in San
Diego, CA. In addition, two sera from apparently healthy hu-
mans, two sera from healthy dolphins, and two sera from
healthy laboratory mice were used as negative controls.
Lacazia loboi yeast-like cell extracts were obtained from the
footpads of a previously infected BALB/c mouse 6 months
after inoculation, as described by Belone et al. (2). Briefly, L.
loboi yeast-like cells were then centrifuged for 10 min at 3,000
rpm, and the supernatant was discarded. Cells were washed
twice with saline, and the pellet was suspended in 1 ml of
saline. Fungal cells were macerated to a powder with liquid
nitrogen inside a mortar and used in the experiments. The
gp43 protein of P. brasiliensis was extracted and purified per
the method of Taborda et al. (28). Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and Western blot analyses
were done per the methods of Laemmli (14) and Towbin et al.
(29).
* Corresponding author. Mailing address: Biomedical Laboratory
Diagnostics Program, Michigan State University, 322 North Kedzie
Hall, East Lansing, MI 48824-1031. Phone: (517) 353-7800. Fax: (517)
432-2006. E-mail: mendoza9@msu.edu.
Published ahead of print on 24 October 2007.
164
The diagnosis of lacaziosis in the investigated humans and
dolphins was confirmed by clinical and histopathological meth-
ods (Fig. 1). The stained gels of L. loboi protein extracts
showed at least 10 bands, ranging from 193 kDa to 9.0 kDa
(Fig. 2). The L. loboi protein extract lacked a visible protein
band at 43 kDa. The purified gp43 protein of P. brasiliensis
showed a strong band at the expected molecular mass and did
not contain other proteins (Fig. 2). Normal mouse skin tissue
samples processed as described above did not show visible
bands (data not shown).
The immunoglobulin G (IgG) antibodies in the sera of all
tested hosts with lacaziosis detected the gp43 protein of P.
brasiliensis fairly well (Fig. 3). The tested dolphin, human, and
experimentally infected mouse sera detected a major, immu-
nodominant, 193-kDa antigen in the L. loboi protein extract
(Fig. 3) and also reacted weakly with other immunogenic an-
tigens (Fig. 3). The experimentally infected mouse antibodies
mildly detected five other immunogens, including an 48-kDa
protein not detected by the other sera (Fig. 3, panel 2, lane b,
arrowhead). The IgG antibodies of the dolphins detected only
two high-molecular-mass antigens in the L. loboi protein ex-
tract, including the immunodominant, 193-kDa antigen (Fig.
3, panel 3, lane b). The antibodies in the serum from a patient
with paracoccidioidomycosis weakly detected at least 13 other
antigenic components in the L. loboi protein extract, ranging
from 193 kDa to 9.0 kDa. Some of these antigens were not
detected by the antibodies in the sera from humans and dol-
phins with lacaziosis or the antibodies in the sera from exper-
imentally infected mice (Fig. 3, panel 4, lane b). Only the
antibodies in the serum from the patient with paracoccidioid-
omycosis weakly detected a molecular antigen of 43 kDa in the
L. loboi protein extract (Fig. 3, panel 4, lane b). The healthy
FIG. 1. Human parakeloidal (A) and dolphin verrucous (B) lesions
from two of the hosts with lacaziosis used in this study. The histopa-
thology of these lesions is shown in panels C (human) (silver stain;
magnification, 100) and D (dolphin) (silver stain; magnification,
40).
FIG. 2. Coomassie blue-stained sodium dodecyl sulfate-polyacryl-
amide gel showing purified Paracoccidioides brasiliensis gp43 (lane 1)
and the profile of an Lacazia loboi protein extract (lane 2). The figure
shows the 193-kDa immunodominant antigen (arrow) as well as
other proteins in the L. loboi protein extract. The asterisks indicate
common proteins detected by the sera tested in this study. Lane M,
molecular mass marker.
FIG. 3. Western blot analyses of transferred proteins on nylon
membranes. The antibodies in the serum from a human with lacaziosis
weakly detected Paracoccidioides brasiliensis gp43 (lane 1a). This se-
rum strongly detected an 193-kDa immunodominant antigen (arrow)
as well as six other weak bands (asterisks) (lane 1b). The IgG in the
serum from the experimentally infected mouse weakly detected P.
brasiliensis gp43 (lane 2a). The mouse antibodies strongly detected the
193-kDa immunodominant antigen (arrow) as well as seven other
proteins (asterisks) and an 48-kDa protein not detected by the other
sera (lane 2b, arrowhead). The antibodies in the dolphin with lacaziosis
slightly detected P. brasiliensis gp43 (lane 3a). The antibodies strongly
recognized the 193-kDa immunodominant antigen (arrow) and only
one of the minor antigens previously detected in the L. loboi extract
(lane 3b). The antibodies in the serum from the patient with paracoc-
cidioidomycosis strongly detected gp43 (lane 4a). The antibodies of
this patient also detected the immunodominant 193-kDa antigen
(arrow) in the L. loboi extract as well as other antigens (asterisks),
including a 43-kDa antigen (arrowhead) (lane 4b). The molecular mass
marker (lanes M) appears in each of the panels.
V
OL. 15, 2008 NOTES 165
control human, dolphin, and mouse sera did not react with the
antigens used in this study (data not shown). One of the two
control dolphin sera possessed antibodies against the 43-kDa
purified protein of P. brasiliensis and the major antigens of L.
loboi normally detected by the dolphins with lacaziosis. Puta-
tive residual normal mouse skin cells were not detected by the
tested sera (data not shown).
With the exception of three previous studies (1, 23, 30), in
the past 75 years L. loboi immunological research has been
carried out using antigens of P. brasiliensis (6, 8, 15, 17, 21, 24,
26, 27, 32). Our study showed that patients with lacaziosis
possessed antibodies not only against the gp43 antigen of P.
brasiliensis but mainly against an 193-kDa immunodominant
L. loboi antigen. The possibility that the 193-kDa antigen
comprises several compressed L. loboi immunogens is cur-
rently under investigation in our laboratory. The IgGs in the
sera from patients with lacaziosis failed to detect the gp43-like
antigen in the protein extract of L. loboi. However, the serum
from the patient with paracoccidioidomycosis did detect an
antigen with a similar molecular mass. The cross-reactive an-
tigens reported in this study, including purified gp43 and those
reported by other investigators (1, 6, 15, 21, 24, 26, 30), were
not unexpected, since recent molecular studies have shown
that both L. loboi and P. brasiliensis share the same ancestor
(13, 18, 31).
The many reports of lacaziosis in dolphins have puzzled
investigators for decades (3, 5, 9, 12, 19, 22, 25, 32). However,
studies to elucidate its etiology, epidemiology, and immunol-
ogy have been equally challenging (5, 22, 25). Our study
showed that dolphins develop IgG antibodies against similar
prominent antigens to those recognized by humans with laca-
ziosis and by experimentally infected mice. Interestingly, one
of the negative control dolphins detected the gp43 antigen of
P. brasiliensis and the other major antigens of L. loboi identi-
fied by dolphins with lacaziosis. This unexpected result sug-
gests a previous exposure to L. loboi without disease, as it is
frequent among the other dimorphic members of the Onyge-
nales (15, 17, 20). Thus, Western blotting analyses could be a
useful tool for epidemiological studies of L. loboi. Although
some investigators have reported morphological differences
between the yeast-like cells of L. loboi in both species (11), our
study is the first to suggest that dolphins are infected with
similar L. loboi strains to those infecting humans.
In contrast to previous serological reports (1, 23, 30), this
study suggests that during infection, L. loboi presents immu-
nogens to the immune system which are different from that in
P. brasiliensis infection. The molecular characterization of
these antigens, especially the 193-kDa immunodominant an-
tigen, could generate valuable information to better under-
stand the immunology and serology of L. loboi infection and
possibly aid in the development of new therapies for infections
caused by this resilient fungal pathogen.
Lesions from the free-ranging dolphin were collected under Na-
tional Marine Fisheries Service scientific research permit no. 998-1678,
issued to Gregory D. Bossart as part of the Bottlenose Dolphin Health
and Risk Assessment (HERA) Project conducted in the Indian River
Lagoon, FL, and the estuarine waters of Charleston, SC. Samples from
the stranded dolphin were collected under a National Marine Fisheries
Service letter of agreement, and this collection was supported in part
under award NA06NMF4390138 from the National Oceanic and At-
mospheric Administration, U.S. Department of Commerce. This re-
search was supported in part by the Biomedical Laboratory Diagnosis
Program, Michigan State University, MI.
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VOL. 15, 2008 NOTES 167
... Studies using serum samples from humans and dolphins with lacaziosis/lobomycosis, mice experimentally infected with L. loboi, and serum samples from humans with paracocidioidomycosis showed that IgG in serum samples from dolphins and humans infected with L. loboi had strong cross-reactivity with the gp43 antigen of P. brasiliensis (4,6,21). These findings support the hypothesis that the uncultivated organism causing cutaneous granulomas in humans and dolphins was L. loboi. ...
... Findings also implied that the gp43 antigen of the etiologic agent of parakeloidallike granulomas in humans and dolphins was antigenically similar to that of P. brasiliensis. On the basis of these serologic studies (4,6,21), current phylogenetic data for gp43 and Kex gene exons, and ITS DNA sequences, placement of L. loboi from humans in its own genus is questionable. Efforts to culture the organism from dolphins on classical laboratory media successfully used to isolate P. brasiliensis from humans with paracoccidioidomycosis were not successful (4,7). ...
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2097 Cutaneous granulomas in dolphins were believed to be caused by Lacazia loboi, which also causes a similar disease in humans. This hypothesis was recently challenged by reports that fungal DNA sequences from dolphins grouped this pathogen with Paracoccidioides brasiliensis. We conducted phylogenetic analysis of fungi from 6 bottle-nose dolphins (Tursiops truncatus) with cutaneous granu-lomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasil-iensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidi-oidomycosis ceti.
... No serological tests are currently available for commercial use.Western blot analysis and PCR techniques have been developed. 110 A fluorescein diacetateethidium bromide (FDEB) staining technique has been found to be reliably sensitive and specific in determining viability of L. loboi to measure therapeutic success. 110,111 Treatment Wide surgical excision ensuring that margins are free of infection, remains the most successful treatment. ...
... 110 A fluorescein diacetateethidium bromide (FDEB) staining technique has been found to be reliably sensitive and specific in determining viability of L. loboi to measure therapeutic success. 110,111 Treatment Wide surgical excision ensuring that margins are free of infection, remains the most successful treatment. Electrodessication or cryosurgery is useful for early small lesions. ...
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... 57 In contrast, serum from an infected dolphin recognized an immunodominant 193-kDa antigen from an extract of human L. loboi more strongly than the gp43 antigen of P. brasiliensis in western blotting analyses. 47 Experimental inoculation of a laboratory scientist with yeast-like cells from a human patient, as well as a report of an accidental transmission of lobomycosis in another laboratory scientist who collected and purified fungal cells from human skin biopsies, 50 imply that under unusual circumstances, Lacazia of human origin can be transmitted to other humans. 24 The epidemiologic data of lobomycosis in endemic areas is unknown. ...
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... One study demonstrated that individuals with lacaziosis possess antibodies reactive to the gp43 antigen of P. brasiliensis, and also to a 193 kDa major L. loboi antigen through WB. The cross-reactivity occurs because, as supported by molecular studies, L. loboi and P. brasiliensis share a similar ancestor [187]. In contrast to prior reports, this study proposes that, during infection, L. loboi presents antigens that are distinct from that presented during paracoccidioidomycosis [188,189]. ...
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... In humans, lobomycosis acts as a chronic fungal skin infection that mainly occurs in rural areas of South and Central America, where this disease is endemic (Brito and Quaresma 2007 ). According to Haubold et al. ( 2000 ) the etiologic agents responsible for lobomycosis in humans and dolphins are not the same; notwithstanding, evidence from serologic data suggests that humans and dolphins are infected by the same L. loboi strains (Mendoza et al. 2008 ). In dolphins, lobomycosis is distributed from southern Brazil to the Gulf of Mexico and Atlantic coast of Florida (Reif et al. 2006 ). ...
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